Study On The Effect Of Melatonin On Quality Of Frozen-Thawed Sperm In Huang-Huai Sheep Based On Proteomic And Metabolomic Analyses

Huang-huai sheep is a new multiparous mutton breed that has been cultivated by domestic scientific research institutes,governments,and sheep farms in China.This breed was bred using binary cross breeding with introduced Dorper sheep as a sire and native Small-Tailed Han sheep as a dam.This variety has high mutton quality and high fecundity and is only distributed in partial regions of central China plain.Cryopreservation approach can store and transport ram spermatozoa for a long time,which is beneficial to the development of artificial insemination,in vitro fertilization and other reproductive technologies.However,freezing and thawing processes force spermatozoa to osmotic damage,oxidative stress,etc.,together with the difference of breed(individual)or treatment ways,thereby causing the difference of sperm fertility change.There are still some technical bottlenecks for the application of ovine sperm cryopreservation in artificial insemination.Exploring the molecular mechanism of frozen-thawed spermatozoa in Huang-huai sheep will provide opportunities for cryopreservation,long-distance transportation and artificial insemination of ram high-quality semen,and promote the sheep production.In this study,Huang-huai sheep were selected as our research object.The motility,antioxidant-related enzymes levels,lipid peroxidation products concentrations,plasma membrane and acrosome integrities of frozen-thawed spermatozoa were evaluated by pretreating and freezing spermatozoa with different doses of the multipotent antioxidant melatonin(MLT).Optimized concentration of the MLT was screened by data differential analyses.Afterwards,the effects of freezing-thawing on proteome and metabolome in spermatozoa were investigated,and the molecular protective effects of the MLT on frozen-thawed spermatozoa were analyzed,based on 4D label-free quantitative proteome combined with UHPLC-QE-MS non-target metabolome techniques.The main results were obtained as follows:(1)Biochemical indices of serum and seminal plasma and the routine parameters of sperm quality were analyzed.The results showed that values of biochemical indices such as ALT,ALP,a-AMY,GLB,TBIL-V,P,TC,TG,FUN in serum were within the theoretical reference range.There were proteins(such as TP,ALB),enzymes(such as LIP,ALP,CREA-SOX,LDH),non-enzymes(such as r-GT,UA,FUN,TG,TBIL-V)and other biochemical factors in seminal plasma,and these factors were correlated with sperm conventional quality parameters.Original sperm motility was more than 80% and the density was more than 55×10^6 /m L.(2)Effects of MLT with different concentrations on motility parameters,antioxidant related enzymes and other indices of frozen-thawed spermatozoa were measured.The results showed that the motility(<45%),movement speed(VCL,VSL,VAP),levels of enzymes(T-AOC,T-SOD,GSH-Px,CAT and Acetyl-Co A),acrosome and plasma membrane integrities were significantly decreased(P <0.05),concentrations of lipid peroxidation products(MDA,4-HNE)were significantly increased(P >0.05)in frozen-thawed sperm compared to fresh sperm.Effects of MLT on the above indices in frozen-thawed sperm were as follows: replenishment of MLT with different concentration gradients(0.1,0.5,1.0,1.5,2.0 m M,respectively)could improve the motility,movement speed(VCL,VSL,VAP),levels of antioxidant enzymes(T-AOC,GSH-Px,CAT),wherein0.5 m M MLT had the best effect(P <0.05).Also,0.5 m M MLT significantly decreased MDA concentration and increased Acetyl-Co A level,acrosome and plasma membrane integrities(P <0.05).(3)Differentially abundant proteins in spermatozoa from different treatment groups(Fresh,NC,MC,respectively)were analyzed using the 4D-label free combined with PRM quantitative proteome technologies.A total of 2,442 proteins were identified,of which1,941 proteins were quantified.With the P <0.05 and FC >1.5,324 differential proteins in MC vs.Fresh,266 differential proteins in NC vs.Fresh and 10 differential proteins in MC vs.NC were screened,respectively.GO and KEGG enrichment analysis indicated membrane lipolysis and metabolism,glucose and lipid metabolism,microtubule cytoskeleton organization,cellular protein-containing complex assembly,electron respiratory transport chain,OXPHOS,flagellar organization,plasma membrane bounded cell projection assembly,apoptosis,pentose and glucuronate interconversions,AMPK signaling and m TOR signaling pathways were significantly enriched.Among which the differential proteins such as CST,PA200,AKR1B1,COX6 C,Zm QCR7,LOC101117965,HBB,SOD1,PARK7,etc.may exert regulated role.(4)The abundance changes of metabolites in frozen-thawed sperm were analyzed using UHPLC-QE-MS non-target metabolome technology.A total of 597 metabolites mapping MS2 names were identified in ram sperm,including 439 in POS mode and 158 in NEG mode.Freezing stimulation significantly affected the abundance of 376 metabolites,with191 up-regulated and 185 down-regulated.MLT induced changes in the abundance of 25metabolites(20 up-regulated and 5 down-regulated)in frozen-thawed sperm(P <0.05,VIP >1).Combined with functional enrichment analysis,MLT reversed abundance of the differential metabolites in frozen-thawed sperm,which including up-regulated metabolites such as N-acetyl-5-methoxy tryptamine,acetyl-N-formyl-5-methoxykynurenamine,indole-3-carboxylic acid,2-formamido-benzoic acid,prostaglandin E2,L-glutamic acid and down-regulated metabolites such as acetylcholine.They were involved in molecular pathways related to sperm anti-oxidative stress,motility,scavenging of free radical,DNA integrity,etc.(5)Based on mix Omics R package analysis,29 differential proteins and 26 differential metabolites closely associated with the function were focused in frozen-thawed sperm.Simultaneously,MLT may protect post-thawed sperm through reducing oxidative stress,maintaining OXPHOS and microtubule structure.Potential molecular markers such as PRCP,NDUFB8,NDUFB9,SDHC,EIF6,DCTN1,TUBB6,TUBA3 E,SSNA1 proteins,L-histidine,L-targinine,ursolic acid,xanthine metabolites were captured,those are used to assess the quality of freezing-thawing ram sperm and are worth exploring in depth.In conclusion,this study demonstrated that 0.5 mM MLT can improve the motility,antioxidant enzymes and Acetyl-Co A levels,plasma membrane and acrosome integrities,and reduce lipid peroxidation of frozen-thawed spermatozoa.Simultaneously,the study also proved the effects of MLT on proteome and metabolome of frozen-thawed spermatozoa in Huang-huai sheep,found potential pathways and molecular markers.Which laid a foundation for further research on the molecular mechanism of MLT protection spermatozoa against freezing-thawing damages,improving cryopreservation of high-quality ram semen and promoting the process of artificial insemination for sheep.